(1) Field of the Invention
The present invention relates generally to poultry protection, in particular to a Mycoplasma gallisepticum (MG) protective formulation.
(2) Description of the Prior Art
Mycoplasma gallisepticum, a pathogen of the avian respiratory tract, belongs to the class Mollicutes, order Mycoplasmatales, and family Mycoplasmataceae. Under certain circumstances, MG may be associated with acute respiratory disease in chickens and turkeys, especially in young birds, with the turkey being more susceptible. It can also cause upper respiratory disease in game birds. Edematous changes early in infection indicate an immunopathological response may be responsible for some of the pathology observed. More recently it has been recognized as a cause of conjunctivitis in house finches in North America. The severity of the disease is greatly affected by the degree of secondary infection with viruses such as Newcastle disease and infectious bronchitis, and/or bacteria such as Escherichia coli. A more chronic form of the disease may occur and can cause reduced egg production in breeders and layers. The infection is spread vertically through infected eggs and horizontally by close contact. Other methods of spread are less well documented. MG can be detected in two ways. The presence of MG can be confirmed by isolating the organism in a cell-free medium or by detecting its DNA directly in infected tissues or swab samples. When results are equivocal, chicken embryos or chickens may be inoculated with suspect material. Infection is diagnosed by demonstration of the organism or its DNA or by the detection of specific humoral antibodies.
Samples for isolation consist of swabs of organs or tissues, exudates or diluted tissue homogenates. Culture may also be attempted from aspirates from the infraorbital sinuses or joint cavities and from yolk or embryos. The sample selected will be influenced by the clinical signs and by any lesions present. Broth and agar are used for isolation, but it is normally necessary to obtain mycoplasma colonies on agar before attempting identification. If there is difficulty, an initial passage of the material through mycoplasma-free chicken embryos or mycoplasma-free chickens may be helpful.
Although some basic biochemical tests can be helpful in preliminary classification of mycoplasma isolates, final identification must be by serological or DNA tests. The most satisfactory of these are PCR, the fluorescent antibody test, and the immunoperoxidase test.
DNA detection methods, mainly based on the polymerase chain reaction, have come into use in specialized laboratories. A commercial kit has been marketed in several countries and can be used to detect field strains and to distinguish them from the vaccine F strain.
Several serological tests have been used to detect MG antibodies, but specificity and sensitivity have been lacking to some degree in all of them. They are more satisfactory for flock screening than for testing individual birds. The most commonly used are the rapid serum agglutination (RSA) test, the enzyme-linked immunosorbent assay (ELISA) and the hemagglutination inhibition (HI) test. In the RSA test, sera from individual birds are tested for agglutination using commercially produced stained MG antigen. Chicken or turkey sera that yield agglutination reactions with the antigen within 2 minutes should be heated at 56° C. for 30 minutes and retested. Sera that still react, especially when diluted, are considered to be positive. These may then be confirmed by an ELISA or a HI test.
Mycoplasma gallisepticum (MG) can cause significant economic loss in chickens and turkeys due to chronic respiratory disease, downgrading of carcasses in meat-type birds and loss of production in layers. Sporadic outbreaks, having important economic consequences, occur in turkeys and broiler chickens, and remain a costly enzootic infection in commercial egg layers.
Although the preferred method of MG control is maintenance of MG-free flocks, both live and inactivated vaccines have been used.
Vaccines can, in principle, be divided into two groups, i.e. live (attenuated) vaccines and inactivated vaccines. The advantages of live vaccines include the presentation of all the relevant immunogenic determinants of an infectious agent in its natural form to the host's immune system, and the necessity of a relatively small amount of the immunizing agent, because of its inherent property to multiply in the vaccinated host. A major disadvantage of a live vaccine is a concern with its safety: a live vaccine may induce disease (in immuno-compromised) animals, or the live microorganism may even revert to virulence, as a result of which animals experience a virulent infection.
The disadvantage concerning the safety aspect is not displayed by inactivated vaccines and, hence, constitutes the major advantage over live vaccines. However, a major disadvantage of inactivated vaccines is represented by their intrinsically low immunogenicity, i.e. inactivated immunogens as such have a limited ability to trigger the host's immune system. Therefore, appropriate means are necessary to augment the immunogenicity of these inactivated immunogens. This type of vaccine normally requires adjuvants with significant immunostimulatory capabilities to reach a minimum potential in preventing disease. Thus, the desirability of additional immunostimulators for use in combination with inactivated immunogens in order to augment their inherently low immunogenicity is evident, in particular immunostimulators which are applicable to more than one immunogen.
MG vaccines represent an unusual situation in that they are not recognized for general widespread use in poultry. They are generally used in birds of multiple-aged egg-layer sites to prevent egg production losses caused by wild-type strains. This is important because once the MG infection is present in a flock it remains permanently. Currently ⅔ of commercial layers are vaccinated in the United States. Internationally, there is high demand for live vaccines. In countries such as Australia the entire broiler breeder replacement stock is vaccinated using live vaccines.
In current practice, prophylactic immunization of fowl against MG related disease involves either controlled exposure to attenuated vaccine strains, or the use of inactivated, whole cell oil emulsion vaccines. Each of these approaches has certain disadvantages. For example, live vaccines can produce disease or impair reproductive function. Inactivated vaccines, while generally effective in preventing disease in immunized birds, do not reliably prevent infection, and may allow spread of infection and disease to unvaccinated birds.
In the US, vaccination is generally used only on multi-age sites where it is inevitable that flocks will become infected. The usual purpose of vaccination is to prevent egg production losses in commercial layers, although vaccines may also be used to reduce the level of egg transmission in breeding stock or as a tool for MG eradication on multi-age production sites. It is important for either live or killed vaccine to be administered before the flock is exposed to field infection with MG.
Available live vaccines are generally produced from the F strain of MG, and, more recently, strains ts-11 and 6/85, which are nonpathogenic strains with improved safety characteristics. Administration of the F strain by the intranasal or eyedrop route is preferred, but aerosol or drinking water administration may be used. The eyedrop method is recommended forts-11, while a fine spray is recommended for 6/85. Pullets are generally vaccinated between 12 and 16 weeks of age. A single dose is sufficient and vaccinated birds remain permanent carriers. Long-term use of the F strain on a multi-age site results in displacement of the field strain with the vaccine strain. F strain displaces virulent wild-type MG strains more efficiently than 6/85 or ts-11, but ts-11 has been successfully used to eradicate F strain MG in multi-age commercial layers. Multi-age production sites are also known to test serologically negative for MG after long-term use of 6/85. The F strain is fully virulent for turkeys.
U.S. Pat. No. 5,064,647 issued on Nov. 12, 1991, to Paul K. Storm is directed to a Mycoplasma vaccine based on the 6/85 strain. The 6/85 strain is attenuated by serial passage in media. For chickens it is extremely attenuated. While 6/85 may be used effectively in chickens it does not retain its attenuated state in turkeys.
Ts-11, on the other hand, is a temperature-sensitive mutant that has been chemically mutagenized. It is used universally in Australia, Latin America and Asia. It is also used in ⅔ of the United States market. Like 6/85, ts-11 is useful for vaccination of chickens against MG but not useful for vaccinating turkeys.
Current live MG vaccines are thus problematic. F-strain is virulent in turkeys. And attenuation of both 6/85 and ts-11 is unstable because the vaccine strains have unstable genomes that revert to virulent disease-causing wild-type. But, over time the turkeys outgrow the vaccines and the virulent field MG infection takes over. As such current live MG vaccines are not permitted in states such as North Carolina and Minnesota with large turkey populations. The country of Israel, as well, disallows the use of live MG vaccines.
Also, the 6/85 and ts-11 strains do not persist in chickens. In fact, prior art teaches away from persistence as a protective mechanism. U.S. Pat. No. 5,064,647 issued to Paul K Storm on Nov. 12, 1991 teaches that a benefit of strain 685 is that the strain is no longer detectable in chickens 4 weeks after inoculation. Eventually the wild-type strain returns causing disease and loss of production. On the other hand, less attenuated live vaccine strains do not adequately prevent the disease. Thus there appears to be an inverse relationship between persistence of the vaccination and mildness of the disease.
An alternative to vaccination with live organisms for preventing MG is the use of bacterins. Bacterins consist of a concentrated suspension of MG organisms in an oil emulsion. These are ordinarily administered to growing pullets at 12-16 weeks of age. They are administered parenterally, usually subcutaneously in the neck. Although two doses are desirable, a single dose is usually given because of cost and labor considerations. Bacterins are effective in preventing egg-production losses and respiratory disease, but they do not prevent infection with wild-type MG.
Thus, there remains a need for a safe and efficacious formulation to protect birds susceptible to MG. Optimally the formulation should include a live strain that persists yet causes minimal pathology in both chickens and turkeys. The protective formulation should also be stable and non-virulent in turkeys. The present invention solves this longstanding need.